Patent Application
20210001009
The present invention relates to the emerging fields of 3D bioprinting and functional tissue engineering. More specifically, embodiments of the invention relate to compositions which include biogums and/or botanical gums in combination with a biocompatible biomaterial to constitute a bioink capable of use in bioprinting of mammalian and human tissue constructs for subsequent use in in vitro culture, transplantation, tissue development, and drug screening and development.
In three-dimensional (3D) printing processes, an object is fabricated layer by layer by a printer device using computer aided design, CAD file. 3D printing has been already successfully used in tissue engineering by many scientists to fabricate patient specific scaffolds. The scaffolds made of thermoplastic polymers have been extruded using 3D printers. The disadvantage of 3D printing using thermoplastic materials is a difficulty in cell seeding due to limited cell migration into porous structures. 3D Bioprinting operates using liquids in room or body temperature and thus can potentially handle living cells. The introduction of 3D Bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine, which can enable the reconstruction of living tissue and organs preferably using the patient's own cells. The 3D bioprinter is a robotic arm able to move in the X,Y,Z directions with a resolution of 10 μm while dispensing fluids. The 3D bioprinter can position several cell types and thus reconstruct the architecture of complex organs. The need for hierarchical assembly of 3D tissues has become increasingly important, considering that new technology is essential for advanced tissue fabrication. 3D cell printing has emerged as a powerful technology to recapitulate the microenvironment of native tissue, allowing for the precise deposition of multiple cells onto the pre-defined position. Parallel to these technological advances, the search for an appropriate bioink that can provide a suitable microenvironment supporting cellular activities has been in the spotlight. Bioinks often include a low viscosity or temperature sensitive biomaterial blended with a thickening agent to impart printability while also preserving cell viability and biological activity.
According to embodiments, biogums such as microbially derived gums (e.g. xanthan gum(s)) or plant-derived (e.g., botanical) are utilized as a thickener in combination with various biomaterials to fabricate ready to print bioinks compatible with a range of printing nozzles and parameters. Embodiments of the invention rely on the discovery that the combination of two polymers, one a biomaterial-based hydrogel (mammalian, plant based, or microbially derived) or synthetic hydrogel and one a microbial, fungal, or plant based or produced biocompatible polysaccharide which acts as a thickener (e.g., xanthan gum, gellan gum, diutan gum, welan gum, pullalun gum, acacia gum, tara gum, glucomannan, pectin, locust bean gum, guar gum, carrageenan, and tragacanth), with or without cells, for use in the 3D bioprinting of human tissues and scaffolds, results in excellent printability and improved cell function, viability and engraftment.
Embodiments relate to a bioink composition which includes a biocompatible microbial (such as xanthan gum, gellan gum, curdlan gum, welan gum, pullalun gum), fungal, or plant-produced (such as acacia gum, tara gum, glucomannan, pectin, locust bean gum, guar gum, carrageenan, and tragacanth) polysaccharide, with or without cells, together with a mammalian, plant, microbial-derived, or synthetic hydrogel for bioprinting of human tissue analogues and scaffolds under physiological conditions. Furthermore, embodiments of the bioink composition can be supplemented through the addition of auxiliary proteins and other molecules such as extracellular matrix components, Laminins, growth factors including super affinity growth factors and morphogens. The bioink compositions can be used under physiological conditions related to 3D bioprinting parameters which are cytocompatible (e.g., temperature, printing pressure, nozzle size, bioink gelation process). According to one example, the combination of a microbial, fungal, or botanical biogum polysaccharide together with mammalian, plant, microbial or synthetically derived hydrogel exhibited improvement in printability, cell function and viability compared to tissues printed with bioink not containing these biogums. Embodiments thus include products (e.g., human tissue specific bioinks) and methods (e.g., physiological printing conditions), as well as several applications.
In a first aspect, provided is a bioink composition for use in 3D bioprinting comprising:
(i) one or more microbial, fungal, or plant based or produced thickener component (biogum and/or botanical gum),
(ii) one or more mammalian, plant, microbial, or synthetically derived biomaterial component, and
(iii) optionally, one or more auxiliary component,
wherein the bioink composition optionally includes cells.
In some embodiments the composition includes cells, such as human cells.
In some embodiments, the biogum is a xanthan gum produced from Gram negative bacteria of the Xanthomonas genus, including one or more of:
(i) X. campestris
(ii) X. fragaria 1822
(iii) X. arboricola
(iv) X. axonopodis
(v) X. citri
(vi) X. fragaria
(vii) X. gummisudans 2182
(viii) X. juglandis 411
(ix) X. phaseoli 1128
(x) X. vasculorium 702
In some embodiments, the biogum is a gellan gum produced from Gram negative bacteria Sphingomonas eldoda of the Sphingomonas genus.
In some embodiments, the biogum is a Curdlan gum produced from Gram negative bacteria of the Alcaligenes faecalis of the Alcaligenes genus.
In some embodiments, the biogum is a Welan gum produced from Gram negative bacteria of the Alcaligenes genus.
In some embodiments, the biogum is a Pullulan gum produced from the fungus Aureobasidium pullulans.
In some embodiments, the biogum is a botanical gum such as an acacia gum which is produced from plant species, including one or more of:
Acacia nilotica
Acacia Senegal
Vachellia (Acacia) seyal
Combretum, Albizia
In some embodiments, the biogum is a tara gum produced from T. spinos.
In embodiments, the biogum is a glucomannon produced from Amorphophallus konjac.
In embodiments, the biogum is a pectin from rinds of lemons, oranges, apples.
In embodiments, the biogum is a locust bean gum produced from Ceratonia siliqua.
In embodiments, the biogum is a guar gum produced from Cyamopsis tetragonolob.
In some embodiments, the biogum is a carrageenan produced from the Chondrus crispus (Irish moss).
In some embodiments, the biogum is a tragacanth produced from legumes of the genus Astragalus including one or more of:
A. adscendens
A. gummifer
A. brachycalyx
In some embodiments the ratio of xanthan gum or other microbial biogum, such as gellan gum, diutan gum, welan gum, or pullalun gum versus biomaterial by weight (w:w) is in the interval from 5:95 to 95:5 w:w, or from 80:20 to 20:80 w:w, such as 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 w:w, or any range encompassing or including these values.
In some embodiments, the xanthan gum or other microbial biogum, such as gellan gum, diutan gum, welan gum, or pullalun gum thickener component has a concentration in the interval from 0.5 to 20% weight by volume (w/v), including 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20% weight by volume (w/v), or any range encompassing or including these values such as 0.5 to 2% w/v, 2 to 5% w/v, 5 to 8% w/v, 8 to 10% w/v, 3 to 7.5% w/v, 1 to 6% w/v, 4 to 8% w/v, 5 to 15% w/v, 8 to 20% w/v, 2 to 18% w/v, and so on. This concentration level is relevant both as initial and final concentration, and after dilution with other components of the composition.
In some embodiments the ratio of botanical gums versus biomaterial by weight (w:w) is in the interval from 5:95 to 95:5 w:w, or from 80:20 to 20:80 w:w, such as 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 w:w, or any range encompassing/including these values.
In some embodiments, the botanical gums thickener component has a concentration in the interval from 0.5 to 50% weight by volume (w/v), including 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45% weight by volume (w/v), or any range encompassing or including these values such as 0.5 to 2% w/v, 0.4 to 1.2% w/v, 0.6 to 1.5% w/v, 2 to 5% w/v, 5 to 8% w/v, 8 to 10% w/v, 3 to 7.5% w/v, 1 to 6% w/v, 4 to 8% w/v, 5 to 50% w/v, 10 to 50% w/v, 10 to 40% w/v, 0.5 to 25% w/v, 20 to 50% w/v, 5 to 45% w/v, 1 to 10% w/v, 5 to 35% w/v, and so on. This concentration level is relevant both as initial and final concentration, and after dilution with other components of the composition.
In some embodiments, the mammalian, plant, microbial or synthetically derived biomaterial is chosen from at least one of the following constituents for cross-linking purposes and/or to contribute to rheological properties of the bioink, such as hydrocolloids or thickening and gelling agents: collagen type I, collagen and its derivatives, gelatin methacryloyl, gelatin and its derivatives, fibrinogen, thrombin, elastin, alginates (such as sodium alginate), agarose and its derivatives, glycosaminoglycans such as hyaluronic acid and its derivatives, chitosan, low and high methoxy pectin, biogums such as gellan gum, diutan gum, glucomannan gum, and/or carrageenans, nanofibrillated cellulose, microfibrillated cellulose, crystalline nanocellulose, carboxymethyl cellulose, methyl and hydroxypropylmethyl cellulose, bacterial nanocellulose, and/or any combination of these constituents.
In some embodiments the concentration of mammalian, plant, microbial or synthetically derived biomaterials is in the interval from 0.5 to 50% (w/v), such as 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45% (w/v), or any range encompassing or including these values, such as from 0.5 to 10% (w/v), and the concentration of cells is in the interval from 0.1 million/ml to 150 million/ml.
In some embodiments, the mammalian, plant, microbial or synthetically derived biomaterials include one or more of:
a. Alginate and its derivatives, such as sodium alginate
b. Agarose and its derivatives
c. Gelatin and its derivatives
d. Collagen and its derivatives
e. Fibrin and its derivatives
f. Hyaluronic acid
g. Basement membrane matrix
h. Laminins
i. Fibronectin and its derivatives
j. Heparan sulfate proteoglycans
k. Cellulose and its derivatives
l. Pectin and its derivatives
m. Chitosan and its derivatives
n. Silk and its derivatives, such as silk fibroin
o. Polyethylene glycol and its derivatives
p. Poly (vinyl alcohol)-based hydrogels
q. Poly(N-isopropylacrylamide) (PNIPAM)
r. Poly(2-hydroxypropyl methacrylate (PHPMA)
s. Poly(2-hydroxyethyl methacrylate) (PHEMA).
In some embodiments, the composition is provided under physiological conditions.
In some embodiments, the composition is provided so that at least one of the following conditions are met:
a. a pH-value for the composition in the interval from 5-8, including 5-7, or from 6-8, or from 7-8, or about 7;
b. the osmolarity of the composition is in the interval from 275 to 300 mOsm/kg, including 275-295, 280-295, 280-300, 285-300 mOsm/kg, such as about 295 mOsm/kg.
In some embodiments, the auxiliary components, such as biomaterials, may be in concentrations ranging from 0.5% to 50% w/v and may include one or more of:
a. Fibronectin and its derivatives
b. Collagen and its derivatives
c. Extracellular matrix
d. Basement membrane matrix
e. Fibrin and its derivatives
f. Elastin and its derivatives
g. Glycosaminoglycans and its derivatives including hyaluronic acid, Chondroitin sulfate, Dermatin sulfate, Heparin sulfate, Keratin sulfate
h. Laminin and its derivatives
i. Small Molecules
j. Peptides (Adhesive, Differentiation, Morphogenic)
k. Lysozyme
l. Growth factors and Morphogens including (a) Proliferative, (b) Differentiation (e.g., Chondrogenic, Fibrogenic, Myogenic, Cardiomyogenic, Neurogenic, Heptagenic, Pancreatic, Renal, Intestine, Dermal, Osteogenic, Oncogenic), and/or (c) Sternness Maintenance (e.g., Chondrogenic, Fibrogenic, Myogenic, Cardiomyogenic, Neurogenic, Heptagenic, Pancreatic, Renal, Intestine, Dermal, Osteogenic, Oncogenic)
m. Fluorescently labeled proteins and biomolecules.
In a second aspect, the invention relates to a method for 3D bioprinting of human tissue comprising bioprinting the composition of the invention, thereby combining a biogum (e.g., microbial gum, botanical gum), and a biomaterial derived from mammalian, plant, microbial or synthetic sources, with human or mammalian cells.
In a third aspect, the invention relates to a method for 3D bioprinting of at least one scaffold comprising bioprinting the composition of the invention, thereby combining a biogum (e.g., microbial gum, botanical gum) based thickener and a mammalian, plant, microbial or synthetic derived biomaterial.
In some embodiments, the method(s) for bioprinting of the invention is/are performed under physiological conditions.
In some embodiments related to the methods for bioprinting of the invention at least one of the following conditions are met during 3D bioprinting:
a. the temperature during the 3D bioprinting is in the interval from 4° C. to 40° C., including 10° C. to 40° C., 20° C. to 40° C., and 30° C. to 40° C., such as 37° C.; or
b. the printing pressure during the 3D bioprinting is in the interval from 1 to 200 kPa, such as below 50 kPa, including 5-45 kPa, 10-35 kPa, and 5-40 kPa, or in the interval from 5-25 kPa when bioprinting with cells.
In a further aspect, the invention relates to a bioprinted tissue or organ prepared by the method for 3D bioprinting with human cells according to invention.
In yet another aspect, the invention relates to the bioprinted tissue or organ according to the invention, for use in therapeutic applications including treatment of liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, skin defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g., liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue.
In another aspect, the invention relates to a method for treating liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, skin defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g., liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue comprising using the bioprinted tissue or organ according to the invention.
In still another aspect, the invention relates to a method for culturing the bioprinted tissue or organ of the invention, wherein the bioprinted tissue or organ is cultured under physiological or pathological conditions.
In some embodiments, at least two types of cells are co-cultured at different ratios. Ratios for cells in co-culture are chosen from: 1:1; 1:5, 1:10, 1:25, 1:50; 1:100, 1:150 and any range in between. In case of more than two cell types in culture the ratio is chosen from: 1:1:1; 1:1:5; 1:1:10; 1:1:50; 1:1:100 and any range in between.
In another embodiment, the method of culturing is for the purpose of in vitro culture, disease modelling, drug screening, biomarker discovery, tissue models for drug development, substance testing and bioactive compound efficacy testing.
In a further aspect the invention relates to an in vitro culture prepared by the method for culturing according to the invention.
The invention also relates to the use of the in vitro culture according to the invention for tissue development, disease development, drug screening and development and biomarkers.
In yet another aspect the invention relates to a bioprinted scaffold prepared by the method for 3D bioprinting according to the invention.
In still another aspect, the invention relates to the use of the bioprinted scaffold according the invention for wound healing.
In a further aspect, the invention relates to a method for preparing recellularised tissue, comprising repopulating the bioprinted scaffold of the invention.
In another aspect the invention relates to a recellularised bioprinted tissue, produced by repopulating the bioprinted scaffold of the invention with human cells.
In yet another aspect the invention relates to a bioprinted tissue, scaffold or recellularised bioprinted tissue of the invention, further comprising growth factors.
In still another aspect, the invention relates to a method for promoting tissue repair, comprising implanting the bioprinted tissue, scaffold or recellularised tissue comprising growth factors of the invention in a diseased tissue or organ.
In another aspect, the invention relates to a method of transplanting a bioprinted tissue, organ or scaffold of the invention, wherein the bioprinted scaffolds and/or tissues are implanted into the diseased tissue or organ, such as ectopically implanted subcutaneously or intra-omentum or directly as tissue-patches into the diseased tissue or organ.
In still another aspect, the invention relates to a method of repairing a tissue or an organ, wherein the bioprinted scaffolds and/or tissues of the invention are implanted as tissue-patches for improving wound healing.
In another aspect, the invention relates to a method of treating a disease in a tissue or an organ, wherein a bioprinted tissue of the invention or a recellularised bioprinted tissue of the invention is applied to the tissue or organ, such as by injection, implantation, encapsulation or extracorporeal application.
In one further aspect, the invention relates to a method for disease modelling, comprising the steps of:
a. providing a bioprinted scaffold of the invention;
b. performing mechanistic investigations; and/or
c. determining the effect of a compound, drug, biological agent, device or therapeutic intervention on the scaffold or tissue.
In still another aspect, the invention relates to a bioprinted tissue, scaffold or recellularised bioprinted tissue for use in one or more of:
a. implantation in a diseased tissue or organ;
b. transplantation into a human or animal body, whereby the bioprinted scaffolds and/or tissues are ectopically implanted subcutaneously or intra-omentum or directly as tissue-patches into the diseased tissue or organ;
c. repairing a tissue or an organ, whereby the bioprinted scaffolds and/or tissues are implanted as tissue-patches for improving wound healing; and/or
d. treating a disease in a tissue or an organ, wherein a bioprinted tissue of claim or a recellularised bioprinted tissue is applied to the tissue or organ, such as by injection, implantation, encapsulation or extracorporeal application.
The accompanying drawings illustrate certain aspects of embodiments of the present invention, and should not be used to limit the invention. Together with the written description the drawings serve to explain certain principles of the invention.
Definition of terms and claim features.
“Biogum” refers to polysaccharides produced by a living organism such as bacteria or other microbials, fungi, or plants; examples of microbial biogums include xanthan gum, gellan gum, diutan gum, welan gum, and pullalun gum.
“Xanthan Gum” refers to a heteropolysaccharide with a primary structure that consists of pentasaccharide units consisting of two mannose, one glucuronic acid, and 2 glucose units. Xanthan consists of a backbone of glucose units with trisaccharide sidechains consisting of Mannose-Glucuronic Acid-Mannose linked to every other glucose unit at the 0-3 position.
“Gellan Gum” refers to a heteropolysaccharide with a primary structure that consists of tetrasaccharide units that consist of two glucose, one glucuronic acid, and one rhamnose unit. The backbone structure is glucose-gluruonic acid-glucose-rhamnose.
“Diutan Gum” refers to a polysaccharide consisting of a repeating unit that is composed of a six sugars. The backbone is made up of d-glucose, d-glucuronic acid, d-glucose, and 1-rhamnose, and the side chain of two 1-rhamnose.
“Welan Gum” refers consists of repeating tetrasaccharide units with single branches of L-mannose or L-rhamnose.
“Pullalun Gum” refers to a neutral polymer composed of α-(1,6)-linked maltotriose residues, which in turn are composed of three glucose molecules connected to each other by an α-(1,4) glycosidic bond.
“Botanical gum” refers to polysaccharide biogums isolated from plants; examples of botanical gums include acacia gum, tara gum, glucomannan, pectin, locust bean gum, guar gum, carrageenan, and tragacanth.
“Acacia Gum” refers to a heteropolysaccharide obtained from the Senegalia (Acacia) senegal and Vachellia (Acacia) seyal trees. This gum contains arabinogalactan which consists of arabinose and galactose monosaccharides that are attached to proteins creating what is known as arabinogalactan proteins.
“Tara Gum” refers to a heteropolysaccharide isolated from T. spinos of the Tara family consisting of a linear main chain of (1-4)-β-D-mannopyranose units attached by (1-6) linkages with α-D-galactopyranose units.
“Glucomannon” refers to a straight-chain polymer, with a small amount of branching isolated from the roots of the konjac plant. The component sugars are β-(1→4)-linked D-mannose and D-glucose in a ratio of 1.6:1.
“Pectin” refers to a heteropolysaccharide found in the primary cell walls of terrestrial plants. These include homogalacturonans are linear chains of α-(1-4)-linked D-galacturonic acid, rhamnogalacturonan II (RG-II), which is a complex and highly branched polysaccharide, amidated pectin, high-ester pectin, and low-ester pectin.
“Locust bean gum” refers to high-molecular-weight hydrocolloidal polysaccharides, composed of galactose and mannose units combined through glycosidic linkages, which may be described chemically as galactomannan. Locust bean gum is dispersible in either hot or cold water, forming a sol having a pH between 5.4 and 7.0, which may be converted to a gel by the addition of small amounts of sodium borate. Locust bean gum is composed of a straight backbone chain of D-mannopyranose units with a side-branching unit of D-galactopyranose having an average of one D-galactopyranose unit branch on every fourth D-mannopyranose unit.
“Guar gum” refers to an exo-polysaccharide composed of the sugars galactose and mannose. The backbone is a linear chain of β 1,4-linked mannose residues to which galactose residues are 1,6-linked at every second mannose, forming short side-branches.
“Carrageenan” refers to a polysaccharides isolated from red algae; carrageenan are high-molecular-weight polysaccharides made up of repeating galactose units and 3,6 anhydrogalactose (3,6-AG), both sulfated and nonsulfated. The units are joined by alternating α-1,3 and β-1,4 glycosidic linkages. Three classes of Carrageenan are Kappa, Iota, and Lambda. Kappa forms stiff gels in the presence of potassium and is isolated from Kappaphycus alvarezii. Iota forms soft gels in the presence of calcium ions and is isolated from Eucheuma denticulatum. Lambda does not gel, and is used as a pure thickener.
“Tragacanth” refers to a dried sap of several species of Middle Eastern legumes of the genus Astragalus, including A. adscendens, A. gummifer, and A. brachycalyx.
“Mammalian, plant, microbial, or synthetic hydrogels” refers to any biocompatible polymer network that exhibits characteristics of a hydrogel. A hydrogel is a polymer network that has hydrophilic (e.g., water binding) properties. Mammalian hydrogels consist of proteins or polymers derived from the various tissues, organs, and cells found in mammals including humans, porcine, bovine. Plant hydrogels consist of proteins or polymers derived from various plants including trees, algae, kelp, seaweed. Microbial hydrogels (also referred to as biogums) include polysaccharides produced by bacteria such as xanthan gum, gellan gum, diutan gum, welan gum, and pullalun gum. Synthetic hydrogels include polymers derived from polyethylene, polyethylene, polycaprolactone, polylactic, polyglycolic acid, and their derivatives.
“Bioprinting” refers to the utilization of 3D printing and 3D printing-like techniques to combine cells, growth factors, and biomaterials to fabricate biomedical parts that maximally imitate natural tissue characteristics. Generally, 3D bioprinting utilizes the layer-by-layer method to deposit materials known as bioinks to create tissue-like structures that are later used in medical and tissue engineering fields.
As used herein, “physiological conditions” include conditions (such as pH, osmolarity, temperature and printing/extrusion pressure) that are typical to the normal living environment for a culture or cells, such as, for human cells, a temperature around 37° C., such as in the interval from 35-39° C., a printing pressure in the interval from 1 kPa to 200 kPa, such as below 25 kPa, a pH in the interval from 5-8, such as about 7, and an osmolarity in the interval from 275 to 300 mOsm/kg, such as about 295 mOsm/kg.
As used herein, “pathological conditions” include exposure of a culture or cells to inflammatory and/or carcinogenic conditions, e.g. recapitulating the disease.
As used herein, “co-culturing” cells means that cells of at least two types are cultured together.
In the context of the present invention, the term “bioprinted scaffold” refers to a bioprinted structure or tissue printed with a composition without cells. On the other hand, the term “bioprinted tissue” refers to a bioprinted structure or tissue printed with a composition with cells. The cells can be autologous, allogeneic or xenogeneic. The cells can be stem cells (e.g., pluripotent, induced pluripotent, multipotent, totipotent; mesenchymal, hematopoietic, embryonic, umbilical cord), primary cells (e.g., primary hepatocytes, primary renal cells), or immortalized cells. The cells can be or include can be or include, for example, cells from tissues such as liver, kidney, heart, lung, gastrointestinal, muscle, skin, bone, cartilage, vascularized tissues, blood vessels, ducts, ear, nose, esophagus, trachea, and eye. These can include endothelial cells, skin cells such as keratinocytes, melanocytes, Langerhans' cells, and Merkel cells, connective tissue cells such as fibroblasts, mast cells, plasma cells, macrophages, adipocytes, and leukocytes, bone tissue cells such as osteoblasts, osteoclasts, osteocytes, and osteoprogenitor (or osteogenic) cells, cartilage cells such as chondrocytes and chondroblasts, muscle cells such as smooth muscle cells, skeletal muscle cells, cardiac muscle cells, any cells having muscle fibers such as type I (slow twitch), type IIa and type IIb (fast twitch), nerve cells such as multipolar neurons, bipolar neurons, unipolar neurons, sensory neurons, interneurons, motor neurons, neurons of the brain (e.g. Golgi cells, Purkinje cells, pyramidal cells), glial cells such as oligodendrocytes, astrocytes, ependymal cells, Schwann cells, microglia, and satellite cells, liver cells such as hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, kidney cells such as glomerulus parietal cells, glomerulus podocytes, proximal tubule brush border cells, Loop of Henle thin segment cells, thick ascending limb cells, kidney distal tubule cells, collecting duct principal cells, collecting duct intercalated cells, and interstitial kidney cells, pancreatic cells such as islets cells, alpha cells, beta cells, delta cells, PP cells, endocrine gland cells such as pancreatic cells, hypothalamus cells, pituitary cells, thyroid cells, parathyroid cells, adrenal cells, pineal body cells, and ovarian cells and testicular cells, exocrine gland cells such as sweat gland cells, salivary gland cells, mammary gland cells, ceruminous gland cells, lacrimal gland cells, sebaceous gland cells, and mucous gland cells, or epithelial cells such as squamous cells, cuboidal cells, and columnar cells arranged in architectures such as simple, stratified, and pseudostratified.
Reference will now be made in detail to various exemplary embodiments of the invention. It is to be understood that the following discussion of exemplary embodiments is not intended as a limitation on the invention. Rather, the following discussion is provided to give the reader a more detailed understanding of certain aspects and features of the invention.
Composition
In a first aspect the invention relates to a bioink composition comprising a biogum-based thickener, and a mammalian, plant, microbial or synthetic derived biomaterial with or without cells depending on the application, with or without auxiliary components.
In embodiments, bioink compositions of the invention can comprise one or more biogum thickener, one or more mammalian, plant, microbial or synthetic biomaterial, and one or more auxiliary components.
The biogums can be derived from different mechanical, enzymatic and/or chemical steps known in the art which are performed on the source material (e.g., plant based (or botanical), fungal, or microbial). The bioink compositions or components are typically prepared using sterile components and prepared in clean room conditions.
In embodiments, the bioink composition can include one or more buffer such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N′-bi s(2-ethanesulfonic acid)), TES (2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino] ethane sulfonic acid, N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and CAPS (N-cyclohexyl-3-aminopropanesulfonic acid). The bioink composition can also include one or more solvent such as distilled water, saline, or pH buffered saline. The osmolarity of the composition can be designed to provide compatibility with one or more cell types.
In some embodiments, the composition or one or more of its individual components are provided in desiccated form suitable for reconstitution with a solvent or buffering agent.
Methods for Bioprinting
In another aspect, the invention relates to methods for preparing bioprinted tissues or scaffolds that are suitable for use in the various products, uses and methods of the invention.
In general, the method for 3D bioprinting of human tissue (with cells) or scaffolds (without cells) comprises combining one or more biogum-based bioink, (with or without human cells), and human tissue-specific extracellular matrix (ECM) material, wherein the 3D bioprinting is performed under physiological conditions.
The 3D bioprinted tissue or scaffold can be in the form of a grid, drop, tissue-specific shapes like hepatic lobule for liver etc., or the like. The 3D bioprinted tissue, construct or scaffold can have a printed size in the interval from 0.1 mm to 50 cm in diameter and/or length or width. The bioprinter apparatus can be of any commercially available type, such as the 3D Bioprinters' INKREDIBLE™, INKREDIBLE+™ or BIO X™ from CELLINK AB, or any conventional robotic bioprinter having standard components such as motors, print heads, print bed, substrates for printing, printed structures, cartridges, syringes, platforms, lasers and controls.
Kits for Bioprinting
In one embodiment, the bioink composition is provided in a kit comprising the composition loaded into one or more cartridges, vials, or syringes. The composition can be provided in desiccated form in the kit. The kit can include a separate buffer or solvent for reconstituting the composition, or the composition can be provided already reconstituted with the buffer or solvent already contained in the same cartridge, vial, or syringe as the composition.
A method for preparing bioprinted tissues or scaffolds can be performed under physiological conditions, which could vary depending on the tissue and/or the cells that are printed. Typically, the conditions and parameters during bioprinting varies within the following intervals:
Temperature: 4° C. to 40° C.
Printing pressure: 1-200 kPa.
Also, external cross-linking may be used during or after the bioprinting process such as calcium chloride solution, UV or light exposure in the wavelengths between 300 and 800 nm, such as 365 nm, 405 nm, 425 nm, and 480 nm, or self-assembly of the biomaterial component under thermal incubation. Photoinitiators that can be used include lithium phenyl-2,4,6-trimethylbenzoylphosphinate or LAP. Other photoinitiators can include free radical photoinitiators, cationic photoinitiators, and anionic photoinitiators. The photoinitiator forms a free radical, cation, or anion which subsequently reacts and catalyzes a polymerization or cross-linking reaction. Examples of other photoinitiators include, but are not limited to benzophenone, benzoin-ether, 2-(dimethylamino)ethanol (DMAE), hydroxyacetophenones, 2-hydroxy-2-methyl-1-phenylpropan-1-one and, hydroxyl-phenyl-ketone, Irgacure® 2959, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, (2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-m ethyl-1-propanone; (2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]; 2-isocyanotoethyl methacrylate; benzoyl benzylamine; camphorquinone; thiol-norbornene (thiol-ene); riboflavin; lucirin-TPO; Rose Bengal/furfuryl; ethyl eosin; methacrylic anhydride; 2,2-dimethoxy-2-phenylacetophenone; and Eosin Y.
Bioprinted Tissue or Organ
Other aspects of the invention provide a bioprinted tissue produced by a method described above. Bioprinted tissues produced as described herein display the tissue-specific extracellular matrix protein composition of the source tissue sample.
Bioprinted Scaffold+Use of Bioprinted Scaffold
Another aspect of the invention provides a bioprinted human scaffold or tissue produced as described above for the use in tissue repair, for example.
Bioprinted scaffolds with or without cells and/or with or without known growth factors can be implanted in diseased-tissues or organs, such as tissue-patches, in order to promote tissue repair. For instance, tissue repair can be promoted by wound healing due to the capability of ECM to favor immunomodulation and therefore reducing tissue scarring in fibrotic diseases (e.g. liver fibrosis, intestinal fibrosis, fistulas, Chron's Disease, cartilage defects, etc.).
Methods for Culturing Bioprinted Tissue+In Vitro Culture+Use of In Vitro Culture
Embodiments of the invention provide a bioprinted human scaffold or tissue produced as described above for the use in modeling human diseases, testing drugs and biomarker discovery.
Bioprinted tissue can be used to screen drugs and/or cell-based therapies. For example, bioprinted tissue with cancer cells can be exposed to chemotherapy agents, immunotherapy and/or CAR-T, NK cells.
Methods of Transplantation
Yet another aspect of the invention provides a bioprinted human scaffold or bioprinted human tissue produced as described above for use in the transplantation of a tissue or organ in an individual.
For example, a bioprinted human scaffold or bioprinted human tissue may be transplanted to an individual to replace an organ or a tissue.
Methods of Repairing or Regenerating Tissues and Organs
Another aspect of the invention provides a bioprinted human scaffold or bioprinted human tissue produced as described above for use in the treatment of disease or dysfunction in a tissue or organ in an individual.
For example, a bioprinted human scaffold or bioprinted human tissue may be implanted in an individual to regenerate a complete new organ or to improve the repair of a damaged organ, or may support the organ function of the individual from outside the body.
Methods of Treating a Disease
The bioprinted scaffold or tissue may be useful in therapy, for example for the replacement or supplementation of tissue in an individual.
A method of treatment of a disease may comprise implanting a bioprinted human scaffold or bioprinted human tissue produced as described above into an individual in need thereof.
The implanted bioprinted scaffold or tissue may replace or supplement the existing tissue in the individual.
The bioprinted scaffold or tissue may be used for the treatment of any one of the diseases chosen from, but not limited to: liver diseases, metabolic diseases, diabetes, heart diseases, kidney diseases, lung disease, skin defects, muscle defects, bone defects, bone and soft tissue sarcomas, lung diseases, vessels repair, intestinal diseases, fistulas, cartilage defects, retinal defects, bladder diseases, prostate diseases, tissue fibrosis (e.g. liver, kidney, intestine, lung, skin), cancer in any tissue, such as hepatocellular carcinoma, metastases in any tissue, such as the liver, colon or pancreas, colon cancer, lung cancer, liver cancer, pancreatic cancer, and cancer in any other tissue disclosed in this application, comprising using the bioprinted tissue, organ or scaffold.
Methods for Disease Modelling
The bioprinted tissue or bioprinted scaffold may be useful for disease modelling. Suitable ECM source(s) may be derived from a normal tissue sample or pathological tissue sample, as described above.
A method of disease modelling may comprise:
providing a bioprinted tissue or scaffold produced as described above, optionally bioprinting the tissue or scaffold with cells to produce a recellularised bioprinted tissue, and determining the effect of a compound, drug, biological agent, device or therapeutic intervention on the bioprinted scaffold or tissue or the cells therein.
Methods described herein may be useful in modelling tissue diseases or diseases affecting the tissue, such as tissue fibrosis, tissue cancer and metastases, tissue drug toxicity, post-transplant immune responses, and autoimmune diseases.
Methods of Diagnosis
Bioprinted scaffolds and tissues may be useful for the diagnosis of disease. Suitable bioprinted scaffolds and tissues may be derived from tissue from an individual suspected of having a disease in the tissue or organ.
A method of diagnosing disease in a human individual may comprise: providing a bioprinted scaffold or tissues from the individual produced as described above, and determining the presence and amount of one or more scaffold proteins in the sample.
The presence and amount of scaffold proteins in the sample may be indicative of the presence of disease in the tissue or organ of the individual.
Other Applications
The bioprinted scaffolds and tissues may also be useful for proteomics, biomarker discovery, and diagnostic applications. For example, the effect of a protease on the components, architecture or morphology of a bioprinted scaffold and tissue may be useful in the identification of biomarkers.
The invention will now be described by the following non-limiting examples:
Examples 1-3 provide rheological data for a bioink composition of the invention (GelXG). The bioink composition GelXG comprises: 5% GelMA (Gelatin Methacryloyl)+1.5% Xanthan Gum+LAP 0.25% (lithium phenyl-2,4,6-trimethylbenzoylphosphinate)+2.3% mannitol+10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) as a buffer. Example 4 provides rheological data for another bioink composition of the invention (SilkInk). The bioink composition SilkInk comprises: 15% w/v silk fibroin+1% w/v Alginate (e.g., sodium alginate)+10% w/v Xanthan Gum as thickener. Example 5 provides rheological data for another bioink composition of the invention (chitosan-glucomannan bioink), which bioink composition comprises 3.18% w/v chitosan, 1.818% w/v glycerol phosphate disodium salt (GP), and glucomannan (GM) in an amount of 0.909% w/v. These examples are merely illustrative and should not be construed as limiting any particular feature of the invention.
The test was performed using a 20 mm plate-plate geometry (Discovery Hybrid Rheometer 2, TA instruments, UK), starting at 33° C. and finishing at 15° C. The test is run at a constant angular frequency of 10 rad/s. Average values, from two replicates, of the storage modulus G′, loss modulus G″ and tan 8 are presented in
The test was performed using a 20 mm plate-plate geometry (Discovery Hybrid Rheometer 2, TA instruments, UK). The flow sweep was performed at four temperatures: 20° C., 26° C., 30° C. and 37° C., at shear rates ranging from 0.002 s−1 to 500 s 1. The flow sweeps are compared in
The tests were performed using an 8 mm serrated plate-plate geometry (Discovery Hybrid Rheometer 2, TA instruments, UK). A frequency sweep was performed between 0.16 Hz and 6.3 Hz, the storage modulus, loss modulus and complex viscosity were plotted. Thereafter, an amplitude sweep at a frequency from the linear region of the storage modulus was performed at the same sample. All tests were performed at 20° C., on 3D printed samples (diameter=8 mm, height=2 mm) which, had been cross-linked with UV (405 nm) for 20 s.
Preparation: A first solution (30% w/v silk fibroin (SF) solution) and a second solution (Alginate (e.g., sodium alginate) xanthan gum (XG) blend) were mixed together using a Luer lock adapter in 1:1 ratio between the syringes by moving them back and forth up to 10 times to result in the final concentration of components (15% w/v silk fibroin+1% w/v Alginate (sodium alginate)+10% w/v Xanthan Gum). Three batches were prepared in total. The first batch apparently had some silk self-assembly after mixing, while the other two were mixed even more gently to minimize this effect.
The present invention has been described with reference to particular embodiments having various features. In light of the disclosure provided above, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on the requirements and specifications of a given application or design. When an embodiment refers to “comprising” certain features, it is to be understood that the embodiments can alternatively “consist of” or “consist essentially of” any one or more of the features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.
It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the invention. Further, all of the references cited in this disclosure are each individually incorporated by reference herein in their entireties and as such are intended to provide an efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing the level of ordinary skill in the art.
This application relies on the disclosure of and claims priority to and the benefit of the filing date of U.S. Provisional Patent Application Nos. 62/750,390 and 62/750,417, filed Oct. 25, 2018, each of which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/58025 | 10/25/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62750390 | Oct 2018 | US | |
| 62750417 | Oct 2018 | US |
